FUNSTAR CAPSULE Résumé de rapport

A major focus of modern healthcare has been providing effective and inexpensive treatment to combat diseases. While therapies based on drugs remain the frontline treatment for different diseases including cancer, the rising cost of therapeutic drugs coupled with the complications from improper dosages impart huge financial burden on the economy of a country. The high cost of drugs significantly arises from the development process, where failure is more common. According to the US Food and Drug Administration, only ~8% of the drug candidates that enter clinical trial after initial screening are eventually approved. Therefore, identification of many lead candidates in early discovery stages would increase the success rate of effective drug discovery. However, traditional screening methods are often very slow and use sophisticated instruments, restricting the possibility of rapid drug development. In this project, we introduce novel methodologies for screening drug candidates using colorimetric readout, resulting in quick screening. This approach exploits the the aggregation state of gold nanoparticles (NPs) that is chemically tuned. During the project period, we have successfully created a generic platform for rapid screening of drug candidates for important protein targets related to cancer, epilepsy, glaucoma, and AIDS. Further use of the nanoparticle aggregation process has enabled detection of inhibitors of sphingomyelinase (SMase), an enzyme related to cardiovascular and Niemann-Pick diseases. The significant results obtained during this Marie Curie IIF fellowship can be summarized into the following key novelties: 1) Synthesis of ligands and NPs 2) Fabrication of plasmonic nanostar-stabilized oil-in-water emulsions with the ability to detect small molecules3) Screening of library of compounds for different protein targets4) Generation of SMase-responsive liposomes for colorimetric screening of inhibitors

A detailed progress towards the objectives for each task can be described as follows:1. Synthesis of ligands and NPs was central to the development of the project components. Appropriate organic ligands with target binding ability were designed and synthesized by standard organic synthesis. Likewise, peptide ligands were designed for a particular target and were synthesized through solid phase peptide synthesis. All the ligands were characterized by the standard spectroscopic and chromatographic techniques. The ligands were synthesized with significantly high yield, indicating easy scale-up for large production. The ligands showed good stability in the detection buffers. Successful synthesis of the ligands containing the colorigenic tag shows the possibility of using the screening strategy as a generic drug development methodology for any kind of disease targets. We synthesized gold NPs and modified the conditions to make them stable in the experimental buffer conditions. Prof. Luis Liz-Marzan’s group collaboratively provided gold nanostars (NS) that showed the characteristic plasmonic properties.

2. The nanostars were used to generate stable oil-in-water emulsions utilizing non-covalent bonding between the NS and the oil core. We investigated the synergistic effect of two supramolecular assemblies at the oil-water interface that would provide higher stability of the nanoemulsions. First, the oil core of the capsule was engineered to provide nanoscale assembly of NS at the interface. We exploited a second supramolecular assembly of the coated NS and a complementary compound that offered lateral stabilization of the emulsion shell. By employing this combined supramolecular approaches, we have fabricated functional NS-stabilized capsules that possess numerous plasmonic hotspots for interacting with analytes of interest. While the capsule platform detects thiol-containing small molecules using Raman spectroscopy, the resolution was low and even reduced for biomolecules. However, the nanoemulsions render pH sensitivity, demonstrating a great potential for applications in other applications including drug delivery into cancer cells.

3. Rapid screening of drug candidates – the primary goal of the fellowship – was achieved using gold NP-based colorimetric methodology. We have developed for the first time a smart strategy using synthetic probes and an enzyme to rapidly (within minutes) identify effective drug candidates for different targets. The probes have been designed for specific disease-related target proteins, containing a binder and a colorigenic tag. The tag is easily attached to various types of ligands, leading to a generic platform for diverse disease targets. The methodology has been carefully established for three protein targets, dysregulation of which cause diseases. Upon synthesis of the designed ligands, their binding kinetics and thermodynamics were characterized by surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and UV-Vis spectroscopy that showed significant binding strength. Effective drugs with higher binding affinity towards the target protein were observed to produce colour change. Inhibitory concentrations of the drug candidates matched the binding affinities very well, suggesting the robustness of the method. Taken together, this nanoparticle-based rapid, efficient, and inexpensive drug screening platform shows great potential in expediting drug discovery and monitoring that has not been achieved before.

4. We developed a new technique to screen inhibitors of SMase enzyme, wherein vesicles were constructed containing a small molecule that causes aggregation of gold NPs and hence colour change. We investigated various parameters to fabricate this new type of liposome platform. The optimized formulation consisting of the natural substrate of the enzyme provided robust vesicles that are stable against the leakage of the encapsulated molecules. The enzyme was demonstrated to disrupt the vesicles with consequent release of the encapsulated molecules that aggregates gold NPs. The methodology allowed detection of significantly low concentrations of SMase by the naked eye that provides a major advance against the current state-of-the-art. Determination of the inhibitory concentrations (IC50) of different inhibitors were achieved using the same colorimetric assay. Notably, the approach has several advantages such as high sensitivity, use of the natural enzymatic substrate, and rapid detection ability that makes the methodology versatile to different targets.

We are preparing three manuscripts based on these significant findings that are likely to be published in high-impact journals. Our manuscripts showcase the first report on (a) a generic platform for high-throughput screening of drug candidates using color responses, (b) vesicle platforms for detecting SMases and their inhibitor drugs by the naked eye, and (c) pH-tunable supramolecular assembly of plasmonic NS on nanocapsule platform. We also published two other articles based on these works (J.H. Soh, Y. Lin, S. Rana, J. Ying, M.M. Stevens, “Colorimetric detection of small molecules in complex matrixes via target-mediated growth of aptamer-functionalized gold nanoparticles" Anal. Chem. 87, 7644-7652 (2015); S. Rana, S.G. Elci, R. Mout, A.K. Singla, M. Yazdani, M. Bendur, A. Bajaj, K. Saha, U.H.F. Bunz, F.R. Jirik, V.M. Rotello, “Ratiometric array of conjugated polymers-fluorescent protein provides a versatile mammalian cell sensor” J. Am. Chem. Soc. 138, 4522-4529 (2016)). Our results are the first step in developing smart systems for detecting biosystems and drug candidates, introducing fast and cost-effective techniques. Application of the present nanochemistry-based methods in assembly and colorimetric detection of bioanalytes show a promising future for the healthcare system, the realization of which will require continued extensive research within this area. The development of rapid and robust drug discovery approaches will hugely strengthen the pharmaceutical and toxicology sectors in Europe. Successful translation of the new methodologies would certainly impact healthcare services by reducing the cost and increasing the effectiveness, and thus offer a strong benefit for the society in general. This Marie Curie Fellowship has been extremely successful, leading to the introduction of novel drug discovery tools and nanocapsule platforms, two high-impact publications and a further three manuscripts in preparation.